The BioForce Nano eNabler 4.6 is a molecular printer that can be used to produce patterned molecular substrates on a variety of surfaces, including 3D surfaces. The nanoscale precision and versatility to deposit practically any biomaterial onto a large variety of surfaces is makes the Nano eNabler unique. In the experiments proposed here, the Nano eNabler will be used to address questions regarding the response of neurons to signaling proteins encountered during development and regeneration. The questions will be approached from different angles as a diverse group of researchers from the fields of cellular neuroscience, stem cell biology with focus on neurodegenerative disorders, and bioengineering/material science will investigate mechanisms of cell differentiation and process and synapse formation in vitro. Ultimately, the results obtained in these studies will be used to design in vivo treatment strategies that lead to improved regeneration in the adult injured or diseased CNS. Experiments of all users will involve the printing of single molecules and complex multicomponent patterns of molecules onto physiological or bioengineered substrates to investigate underlying signaling mechanisms implicated in (1) neuron differentiation and neuronal subtype specification, (2) axon dynamics during development (growth cone motility, axon extension, guidance and branching), (3) dendritic arbor formation, (4) synapse formation and plasticity (using the neuromuscular synapse as a model system), and (5) stem cell/neuron responses to inhibitory molecule combination resembling the adult injured CNS environment. In addition, the Nano eNabler will be used to screen components for incorporation in 3D hydrogel scaffolds that will serve as multifunctional platforms allowing the delivery of cells and therapeutic factors to the injury site. )